New Genes for Nothing, Drug Resistance for Free

Bacteria may seem to live a charmed life. Showing but scant effort, they revise their genomes and acquire antibiotic resistance, prompting some of us to say, “That’s the way they do it.” But do they, really?

As it happens, bacteria can create something from virtually nothing. They can use random, noncoding DNA sequences to generate de novo genes that express antibiotic-hindering peptides.

This possibility is supported by evidence gathered by scientists at Uppsala University. They launched a new study that they hoped would shed light on at least one of the two most likely resistance-promoting mechanisms. In one mechanism, new genes with novel functions arise from existing genes, which may be acquired by horizontal transfer or retrieved from a collection of disused (but modifiable) ancestral genes. In the other mechanism, new genes and proteins evolve from random DNA sequences with no similarity to existing genes and proteins.

Focusing on the latter mechanism, the de novo mechanism, the scientists compiled fully random synthetic expression libraries. Next, they placed random gene sequences on plasmids for expression within bacteria. Finally, they determined whether the expressed peptides conferred any beneficial properties, such as antibiotic resistance.

The scientists, led by medical bacteriologist Dan I. Andersson, PhD, professor in the department of medical biochemistry and microbiology, Uppsala University, identified several short peptides (22 to 25 amino acids long) that could give Escherichia coli bacteria a high degree of resistance to aminoglycosides, an important class of antibiotics used for severe infections. Additional details appeared June 4 in mBio, in an article titled, “De Novo Emergence of Peptides That Confer Antibiotic Resistance.”

“Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold,” the article’s authors wrote. “Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance.”

These findings indicate how the expression of random sequences can spark the origination of new genes. Before these findings, a proper appreciation of the de novo mechanism may have been harder to sustain, particularly since it is probably easier to accept that when a gene already exists, it can be modified and acquire a new function.

“When the project started, we had low expectations,” admitted medicinal biochemist and microbiologist Michael Knopp, PhD, a postdoc in the department of medical biochemistry and microbiology, Uppsala University, and the mBio article’s lead author. “We were amazed when we found peptides able to confer a resistance level 48 times higher.”

The current study, which evaluated some 500 million randomized gene sequences, demonstrates how the evolution of selective benefits may be studied in the future. Many different phenotypes could be explored by expressing highly diverse sequence libraries and subsequently selecting for functions such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources.

“This study is important because it shows that completely random sequences of amino acids can give rise to new, advantageous functions,” Andersson emphasized, “and that this process of de novo evolution can be studied experimentally in the laboratory.”